1. Field of the Invention
This invention is related to the field of synthetic polymers. More specifically, this invention relates to methods, kits and compositions suitable for modulating the solubility of synthetic polymers and, in particular, peptide nucleic acid (PNA) oligomers.
2. Description of the Related Art
Peptides and nucleic acids are naturally occurring compositions which are increasingly utilized in research, diagnostic and therapeutic applications. Though naturally occurring peptides and nucleic acids are generally soluble in aqueous solutions, the solubility of individual compositions of differing sequence can vary substantially, with certain compositions exhibiting little or no solubility in aqueous solution. Additionally, the introduction of products and methods for the synthetic production of peptides and nucleic acids has made available sequence variations which are not known to, or may in fact not, exist in nature. The absence of certain biopolymer sequences in nature may at least partially be due to the limited solubility of the composition.
The limited solubility of certain peptide and nucleic acid oligomers can prohibit what would otherwise be a useful research, diagnostic or therapeutic application for that polymer. Therefore, methods and compositions suitable for improving the solubility of peptides and nucleic acids in aqueous solutions may prove essential to the enablement of new technology which utilizes peptides and nucleic acids which otherwise have little intrinsic water solubility. However, compositions which modulate the solubility of synthetic polymers should preferably be simple and achiral since the effectiveness of complex macromolecules such as nucleic acids and peptides in research, diagnostic or therapeutic applications can be adversely affected by the size, complexity or chirality of attached ligands.
Peptide nucleic acids (PNAs) are non-naturally occurring polyamides (also properly characterized as pseudopeptides) which can hybridize to nucleic acids (DNA and RNA) with sequence specificity. (See: U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571 or 5,786,571 and Egholm et al., Nature 365: 566-568 (1993)). PNAs are candidates for investigation as alternatives/substitutes to nucleic acid probes in probe-based hybridization assays because they exhibit several desirable properties. In preferred embodiments, PNAs are achiral polymers which hybridize to nucleic acids to form hybrids which are more thermodynamically stable than a corresponding nucleic acid/nucleic acid complex (See: Egholm et al., Nature 365: 566-568 (1993)). Being non-naturally occurring molecules, they are not known to be substrates for the enzymes which are known to degrade peptides or nucleic acids. Therefore, PNAs should be stable in biological samples, as well as, have a long shelf-life. Unlike nucleic acid hybridization which is very dependent on ionic strength, the hybridization of a PNA with a nucleic acid is fairly independent of ionic strength and is favored at low ionic strength under conditions which strongly disfavor the hybridization of nucleic acid to nucleic acid (See: Egholm et. al., Nature, p. 567). The effect of ionic strength on the stability and conformation of PNA complexes has been extensively investigated (See: Tomac et al., J. Am. Chem. Soc. 118: 5544-5552 (1996)). Sequence discrimination is more efficient for PNA recognizing DNA than for DNA recognizing DNA (See: Egholm et al., Nature, p. 566). However, the advantages in point mutation discrimination with PNA probes, as compared with DNA probes, in a hybridization assay appears to be somewhat sequence dependent (See: Nielsen et al. Anti-Cancer Drug Design 8: 53-65 (1993) and Weiler et al., Nucl. Acids Res. 25: 2792-2799 (1997)). As an additional advantage, PNAs hybridize to nucleic acid in both a parallel and antiparallel orientation, though the antiparallel orientation is preferred (See: Egholm et al., Nature, p. 566).
PNAs are synthesized by adaptation of standard peptide synthesis procedures in a format which is now commercially available. (For a general review of the preparation of PNA monomers and oligomers please see: Dueholm et al., New J. Chem. 21: 19-31 (1997) or Hyrup et. al., Bioorganic & Med. Chem. 4: 5-23 (1996)). Labeled and unlabeled PNA oligomers can be purchased (See: PerSeptive Biosystems Promotional Literature: BioConcepts, Publication No. NL612, Practical PNA, Review and Practical PNA, Vol 1, Iss. 2) or prepared using the commercially available products.
Limited aqueous solubility and a tendency toward self-aggregation has been a long established and well documented restriction on applications of PNA (See for example: Lee, Morse & Olsvik, Nucleic Acid Amplification Technologies: Application to Disease Diagnositics, Chapter 3 by .O slashed.rum et al., BioTechniques Book Div. of Eaton Publishing (1997) pp. 29-48, at p. 40, lns. 14-26; Corey, D. R., TIBTECH, 15: 224-229, June (1997) at p. 225, col. 1, ln. 37 to col. 2, ln. 2; p. 226, col. 2, lns. 24-30 and p. 229, col. 1, lns. 14-33; Lesnik et al., Nucleosides & Nucleotides, 16: 177-51779 (1997) at p. 1775, lns 1-5; Peyman et al., Angew. Chem. Int. Ed. Engl., 35: 2636-2638 (1996) at p. 2636, col. 1, lns. 13-24; van der Laan et al., Tetrahedron Letters, 37: 7857-7860 (1996) at p. 7857, lns. 1-10; Bergman et al., Tetrahedron Letters, 36: 6823-6826 (1995) and Egholm et al., J. Am. Chem. Soc., 114: 1895-1897 (1992). The solubility properties of PNA oligomers in aqueous solution is known to be very sequence dependent. Purine-rich PNA oligomers are known to be particularly difficult to purify and/or characterize at least partially due to their limited solubility. Similarly, the solubility of PNA tends to decrease as the polymer length increases thereby resulting in a preference for shorter PNAs. Self-aggregation is another property which tends to limit the utility of PNA oligomers. Because certain PNA oligomers cannot be adequately purified or characterized, there are presently a large number of potentially useful PNA sequence variations which are unavailable for evaluation in research, diagnostic or therapeutic applications.
By way of example, the product literature of a commercial vendor of custom PNA oligomers states "For most applications an oligomer of 12-15 is optimal. Longer PNA oligomers, depending on the sequence, tend to aggregate and are difficult to purify and characterize" (See: Guidelines For Sequence Design of PNA Oligomers: PerSeptive Biosystems, Inc. Promotional Literature; 1997-1998 Synthesis Products Catalog, col. 2, lns. 6-11, p. 45). Additionally, this document sets forth several rules for the design of a PNA oligomer which will avoid these limitations. Under the heading "Specific Design Rules" (col. 3, lns. 1-18), the text reads "Length: We will not synthesize any sequences with more than 18 bases, not including linkers, amino acids and labels. Purine Content: Purine rich PNA oligomers tend to aggregate and have low solubility. To avoid that follow these specific guidelines: 1. Of any stretch of 10 bases in the sequence do not have more than 6 purines 2. NO more than 4-5 purines in a row, specifically no more than 3 G's in a row". The vendor suggests that one consider analyzing the other strand if it is otherwise impossible to comply with the limitations set forth in guidelines 1 and 2.
A number of modifications have been made to peptide nucleic acids (PNAs) in order to improve their aqueous solubility or minimize polymer self-aggregation. A commonly used modification of PNA which was first used by the inventors involves the incorporation of one or more positively charged terminal lysine residues (See: Egholm et al., J. Am. Chem. Soc., 114: 1895-1897 (1992) at p. 1896, col. 1, ln. 23 to col. 2, ln. 2). The inventors of PNA, as well as others, have also advocated the preparation of PNAs having backbone modifications which comprise one or more alkyl amine groups (See: U.S. Pat. No. 5,719,262 and Lesnik et al., Nucleosides & Nucleotides, 16: 1775-1779 (1997)). Though these modifications improve aqueous solubility, they also introduce chiral atoms to which are linked moieties having nucleophilic primary amine groups which are positively charged at physiological pH. The introduction of chiral centers into PNA can alter the hybridization properties of the polymer (See: Lee, Morse & Olsvik, Nucleic Acid Amplification Technologies: Application to Disease Diagnositics, Chapter 3 by .O slashed.rum et al., BioTechniques Book Div. of Eaton Publishing (1997) pp. 29-48, at p. 33, ln. 4, to p. 34, ln. 12). Additionally, nucleophilic moieties and particularly primary and secondary amino groups must be protected during synthesis and their presence can complicate synthesis, labeling and/or purification. Though positively charged PNAs may exhibit improved hybridization kinetics (See: Corey et al., J. Am. Chem. Soc., 117: 9373-9374 (1995) and Corey et al., FASEB Journal, 9, A1391 (1995)), positively charged groups may also result in non-nucleobase specific interactions which may lead to increased background in a hybridization-based assays.
Another approach to overcoming the limited solubility and self-aggregation of PNA has been to modify the backbone to incorporate negatively charged phosphate moieties (See: Peyman et al., Angew. Chem. Int. Ed. Engl., 35: 2636-2638 (1996) and van der Laan et al., Tetrahedron Letters, 37: 7857-7860 (1996)). However, one of the most advantageous properties of PNA is the neutral backbone which allows for nucleic acid hybridization which is fairly independent of ionic strength and is favored at low ionic strength under conditions which strongly disfavor the hybridization of nucleic acid to nucleic acid. Backbone modifications which re-introduce a negative charge will likely negate this advantageous property.
Still another approach to overcoming the limited solubility and self-aggregation of PNA has been to prepare PNA-DNA chimeras wherein the negative charge on the DNA part of the chimera reduces the tendency toward self-aggregation and thus improves solubility (See: Uhlmann et al., Angew. Chem. Ed. Engl., 35: 2632-2635 (1996) at p. 2632, col. 2, lns. 33-35). However, PNA-DNA chimeras are segmented molecules which exhibit hybrid properties. For example, the Tm of chimeras examined by Uhlmann et al. were approximately half way between the Tm of the DNA/DNA hybrid and the PNA/DNA hybrid (See: Uhlmann et al., Angew. Chem. Ed. Engl., 35: 2632-2635 (1996) at FIG. 4).
Though not expressly designed or sold to improve PNA solubility, applicants have noted that a commonly used ether-based, achiral hydrophilic straight chain linker (8-amino-3,6-dioxaoctanoic acid) can be used to minimally improve the aqueous solubility of PNA oligomers and particularly PNAs labeled with hydrophobic moieties such as fluorescein and rhodamine dyes. However, the 8-amino-3,6-dioxaoctanoic acid linker moiety is not branched, does not maintain the proper spacing for nucleobase to nucleobase interactions, does not branch from the backbone (typically made part of the backbone) and furthermore, conveys only a very limited improvement in aqueous solubility to the PNA oligomer.
Because the utility of a particular PNA oligomer in a research, diagnostic or therapeutic application will generally be specifically related to its sequence, the above mentioned limitations on sequence diversity may prove to be an Achilles Heel of this newly developed and very promising technology. Therefore, it would be useful to provide methods, kits and compositions suitable for improving the aqueous solubility of PNA oligomers and/or reducing their tendency toward self-aggregation so that a greater number of pure PNA oligomers are available for use in research, diagnostic and therapeutic applications. The preferred methods, kits and compositions will exhibit little or no adverse effects on the hybridization properties or physical characteristics of the PNA oligomer. Thus, the most preferred solubility enhancing modifying moieties will be achiral, non-nucleophilic and uncharged at physiological pH or achiral, non-nucleophilic and positively charged at physiological pH.
Any methods, kits and compositions which enhance the solubility of PNA oligomers, should also be equally useful in improving the solubility of peptides or polyamide and/or reducing or eliminating self-aggregation of the polymer. With certain variations, similar compositions should find utility for the modification of nucleic acids and nucleic acid analogs.